JP4417998B2 - Plant disease control method using Bacillus subtilis strain having antagonistic action - Google Patents

Description

  The present invention relates to a novel Bacillus subtilis strain having antagonistic activity, a microbial preparation for controlling plant diseases containing the same, and biological control of plant diseases using the microbial preparation. On how to do.

  When agricultural chemicals are not used, there is a problem that the yield decreases from 30% to a maximum of 100%. Therefore, the use of agricultural chemicals is essential for increasing the yield. However, abuse of chemically synthesized pesticides causes various problems such as soil, water and agricultural product contamination, toxicity, ecosystem disturbance, and emergence of resistant strains. One method for solving such problems is to develop a biopesticide using antagonistic microorganisms. Biological pesticides are roughly classified into plant extracts, microorganisms, natural enemies, natural physiologically active substances, genetically modified organisms (GMO), and the like. Biological pesticides are safer and more biodegradable than chemically synthesized pesticides and are less expensive to develop.

  Research on biological pesticides, especially microbial fungicides, has been steadily progressing as a major field of plant pathology for over 70 years, and as a result, more than 40 products have been developed over the past 10 years.

  On the other hand, a gram-positive bacterium belonging to the genus Bacillus is a bacterium that has been widely studied and commercialized in the same manner as the genus Pseudomonas. Bacillus bacteria produce biologically active secondary metabolites, forming endospores that are heat-stable, heat-stable, and resistant to harsh environments . Gustafson Inc. has developed a microbial fungicide using Bacillus subtilis strains to treat furrows and cotton and peanut seeds, which is marketed under the trade name Kodiak (Backman et al. Improving Plant Productivity with Rhizosphere Bacteria, pp. 3-8, 1994). In 2001, a microbial disinfectant having a trade name of Bio Yield using a mixture of Bacillus subtilis and Bacillus amyloliquefaciens was marketed. In China, many strains of the genus Bacillus are used to increase yield. Not only that, Bayer Inc. has developed a soil infectious disease control agent containing the Bacillus FZB 24 strain, and US Taensa has issued a microbial fungicide using the same fungus.

  In addition, AgraQuest Inc. of the United States has developed Serenade (trade name), a microbial fungicide using Bacillus subtilis QST713 strain. Unlike other products using Bacillus subtilis strains, this microbial fungicide is effective against more than 40 plant diseases including gray mold, damping-off and powdery mildew. effective. This antagonistic microorganism is known to control various plant diseases through various mechanisms such as competition, parasitism, antibiotic action and induction of resistance, including iturin, plipastatin and surfactin (Surfactin) is an antibiotic containing three groups and produces more than 30 lipopeptides (Ritter, Chemical & Engineering News, 81; 30-35, 2003).

  In Korea, research on the biological control of ginseng root rot was started after harvest since the early 1970s, Fusarium wilt of cucumber and strawberry, Fusarium wilt, sesame blight, and gray mold Studies on the biological control of disease are being conducted. However, there are no successful cases due to instability of effects, difficulty in formulation, low productivity, etc. The first microbial disinfectant in Korea using a Bacillus strain is “Topseed” developed by Greenbiotech Co. Ltd. However, since this top seed is effective only for powdery mildew, there are many restrictions on its use.

  Therefore, the present inventors made an effort to develop a new fungicide exhibiting a strong bactericidal activity against a wide range of plant diseases. As a result, Bacillus subtilis EB120 strain, which is an endophytic bacterium isolated from pepper, was barley udon powder. The present invention is completed by finding that it has an antagonistic action against various plant diseases such as pepper anthracnose, rice blast, tomato gray mold, tomato blight, wheat rust, etc. It came to.

  Accordingly, an object of the present invention is to provide a novel Bacillus subtilis strain having an antagonistic action against plant diseases.

  Another object of the present invention is to provide a microbial preparation for controlling plant diseases comprising the strain.

  Another object of the present invention is to provide a method for controlling plant diseases using the microbial preparation.

  In order to achieve the above object, the present invention provides a Bacillus subtilis EB120 strain (KTCC 10578BP) having an antagonistic action against plant diseases.

  In order to achieve the other object, the present invention provides a microbial preparation for controlling plant diseases comprising the strain or an antagonistic active substance derived therefrom as an active ingredient.

  In order to achieve the above and other objects, the present invention provides a method for controlling plant diseases using the microbial preparation.

Detailed Description of the Invention

  The present invention provides a novel Bacillus subtilis EB120 strain (KCTC 10578BP) that exhibits broad antagonism against plant diseases.

  The novel strain according to the invention is isolated from the pepper leaves. The morphological characteristics, biochemical characteristics of the strain and the results of interpretation of the base sequence of 16S rDNA indicate that the strain belongs to Bacillus subtilis. This new strain is named Bacillus subtilis EB120 and was dated January 6, 2004 under the Budapest Treaty on the International Recognition of Deposits for Microorganisms for Patent Procedures, Korea Institute of Biotechnology (KCTC; Korean Collection) for Type Cultures) (address: Korea Research Institution of Bioscience and Biotechnology (KRIBB), 305-33, Goseong-dong, Daegu-gu, Daejeon, Korea), deposited under the deposit number KCTC 10578BP.

  The Bacillus subtilis EB120 strain according to the present invention exhibits the following morphological and biochemical characteristics. That is, the Bacillus subtilis EB120 strain grows thinly on a trypsin soy agar medium, and in the case of a single colony, it becomes a small snow flower shape (see FIG. 1). Bacillus subtilis EB120 belongs to the genus Bacillus, and shows positive reactions in Gram staining reaction, potassium hydroxide (KOH) test, catalase and oxidase test, and forms thermostable endospores. However, it has a hydrolytic ability for starch and casein. Furthermore, except for maltotriose, the Bacillus subtilis EB120 strain and the ATCC 6051 strain show the same sugar utilization.

  Further, the results of interpretation of the base sequence of 16S rDNA indicate that the Bacillus subtilis EB120 strain according to the present invention has the base sequence of SEQ ID NO: 1 having 99% sequence homology with Bacillus subtilis.

  Bacillus subtilis EB120 strain having such characteristics is widely and widely used for controlling plant diseases including barley powdery mildew and cucumber powdery mildew, red pepper anthracnose, rice blast, tomato gray mold, tomato blight and wheat rust. Shows strong antagonism.

  Therefore, the Bacillus subtilis EB120 strain according to the present invention can be used as a microbial preparation for controlling plant diseases. The strain itself can also be used as such a microbial preparation. The microorganism preparation can be prepared by mixing a culture solution of a strain, a solvent extract obtained therefrom or a single endospore with a carrier, and then forming a powder, pellet, granule, or solution into a dosage form. . Carriers that can be used in the present invention include, but are not limited to, water, white carbon, kaolin, and the like.

  By using the microorganism preparation according to the present invention to treat the soil in which plants are growing or growing plants, it is possible to control plant growth inhibition caused by plant diseases and death caused by this.

The Bacillus subtilis EB120 strain according to the present invention is 1.0 × 10 ⁴ cells / ml to 1.0 × 10 ¹⁰ cells / ml, preferably 1.0 × 10 ⁶ cells / ml to 1.0 × with respect to the target plant. Treated in an amount of 10 ⁹ cells / ml.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Isolation of Bacillus subtilis EB120 strain 5 g of fresh pepper leaves were sterilized by immersing them in 2% NAOCl supplemented with 0.1% Tween 20 for 10 seconds. After 45 ml of sterilized water was added to the leaves thus sterilized, 1 ml was taken with a pestle and mortar, and diluted with distilled water to 1:10, 1: 100, and 1: 1000, respectively. 200 μl of each diluted solution was sprayed on a tryptic soy agar medium containing 40 μg / ml cycloheximide, and then cultured at 30 ° C. until bacterial colonies formed. Colonies were taken from the medium and inoculated into a fresh nutrient agar medium and subcultured 3-4 times to isolate single colonies of bacterial strains.

Example 2: Identification of Bacillus subtilis EB120 strain The bacterial strain isolated in Example 1 was identified using the morphological and biochemical characteristics of the strain and 16S rDNA base sequence interpretation.

(1) Morphological and biochemical characteristics The isolated strain grows thinly on a trypsin soybean agar medium, and in the case of a single colony, it is formed into a small snow flower shape (FIG. 1). The isolated strain was found to belong to the genus Bacillus from positive reaction in Gram staining reaction and potassium hydroxide (KOH) test, positive reaction in catalase test and oxidase test, It also formed endospores and hydrolyzed starch and casein.

In addition, saccharide utilization was investigated using GN2 microplate (supplier: Biolog Inc., USA), and the results are shown in Table 1 below. As a comparative strain, Bacillus subtilis subsp. Subtilis ATCC 6051 strain (purchased from: Genebank of Korea Biotechnology Institute) was used.

  As apparent from Table 1, except for maltotriose, the Bacillus subtilis EB120 strain and the ATCC 6051 strain exhibit the same sugar utilization. That is, in both strains, arbutin, cellobiose, D-fructose, α-D-glucose, D-mannitol, D-mannose (D-mannitol) D-mannose), D-psicose, sucrose, and D-trehalose were all utilized, but a subspecies of Bacillus subtilis (Bacillus subtilis subsp. Subtilis) as a control strain Unlike the isolate of the present invention, ATCC6051 strain also utilized maltotriose.

(2) Interpretation of base sequence of 16S rDNA As a result of interpretation of base sequence of 16S rDNA, it was found that the isolated strain had the base sequence of SEQ ID NO: 1 having 99% sequence homology with that of Bacillus subtilis.

  Such a result proves that the isolated strain is a novel strain of Bacillus subtilis. This new strain was named Bacillus subtilis EB120, and was deposited as a deposit number KCTC 10578BP on January 6, 2004 with the gene bank of the Korea Biotechnology Institute.

Example 3 Assay for Control Activity of Strains Against Plant Disease In order to examine the control activity of Bacillus subtilis EB120 strain (hereinafter referred to as “EB120”) against tomato gray mold, tomato plague, cucumber anthracnose and barley powdery mildew, The target plants were treated with EB120 and inoculated with phytopathogenic fungi 24 hours later.

  Specifically, EB120 was inoculated into a 200 ml sterilized trypsin soy broth medium (Tryptic soy broth, Becton and Dickinson) and cultured in shaking water at 30 ° C. for 3 days at 150 rpm. After the culture broth was diluted with distilled water at a ratio of 1: 3 and 1: 9, 30 ml of the culture broth and dilution were mixed with 100 μg / ml xanthan gum, respectively. Tomatoes, barley, and cucumbers are in plastic pots with 70% of horticultural soil until the 2nd leaf stage, 1st leaf stage, and 2 leaf stage, respectively, and greenhouse conditions (25 ± 5 The target plants were treated with the mixture, dried in the air, and allowed to stand at room temperature for 24 hours.

Subsequently, the induction of the pathogenesis of each pathogen was carried out as follows. In other words, tomato plague and tomato gray mold are migratory suspensions and gray molds extracted from spores (5 × 10 ⁴ spores / ml) of Phytophthora infestans (source: Gangneung University, Korea). (Botrytis cinerea, available from Korea Chemical Research Institute) Each spore suspension (5 × 10 ⁵ spores / ml) is sprayed onto the leaves of tomato seedlings at the 2 leaf stage to cause disease in a constant humidity room at 20 ° C. Induced. Cucumber anthracnose is treated with a spore suspension (10 ⁶ spores / ml) of anthracnose fungus (Colletotrichum orbiculare, source: Agricultural Chemistry Institute) on the leaves of cucumber seedlings at the two-leaf stage. After induction of disease in the room, the disease was caused in a constant temperature and humidity room at 25 ° C. for 3 days. In addition, barley powdery mildew was inoculated with spores of the powdery mildew fungus (Blumeria graminis f. Sp. Hordei, obtained from the Korea Chemical Research Institute) subcultured from the host plant on the leaves of the young barley seedling. The disease was caused in a constant temperature room at 20 ° C.

  As a control group, one treated with 30 ml of xanthan gum solution (100 μg / ml) not containing a strain culture solution was used, and as a comparison group, chlorothalonil (a chemical substance having a controlling effect on each pathogen) chlorothalonil) (Purchased by: Sungbo Chemical Co., Ltd.), Dithianon (Purchased by: Hankook Samgong Co., Ltd.), Dichlorofluanid (Purchased by: Dongbu Hannong Chemical Co., Ltd.) ) And Benomyl (supplier: Dongyang Chemical Industry Co., Ltd.) were used.

The severity of each barley powdery mildew, cucumber anthracnose, tomato gray mold, and tomato plague was measured on the leaves inoculated 7 days, 5 days, 3 days, and 4 days after inoculation of each pathogen. It was investigated by evaluating the lesion area ratio due to sporulation inhibition or the lesion area ratio due to whitening or necrosis. The disease control effect (control value) was calculated based on the following formula 1, and the results are shown in Table 2 below.

In the formula 1, ID _UCG indicates the severity of the untreated control group, and ID _TEG indicates the severity of the treated group.

  As apparent from Table 2, EB120 shows excellent control activity against tomato plague, tomato gray mold and barley powdery mildew.

Example 4: Selection of optimal medium exhibiting excellent antagonism against plant diseases In order to select an optimal medium for production of the EB120 control active substance, culture experiments using various culture media were performed as follows. It was.

EB120 was inoculated into the six types of liquid medium shown in Table 3 below, and then cultured in shaking water at 150 rpm at 30 ° C. for 3 days. Each culture was diluted with distilled water at a ratio of 1: 3 and 1: 9. After adding xanthan gum at a concentration of 100 μg / ml to each diluted solution, barley powdery mildew was induced by the method described in Example 3 and the disease control effect was measured. The results are shown in Table 3 below.

  As apparent from Table 3, it can be seen that the control activity of EB120 against barley powdery mildew is optimal when cultured in a trypsin soybean liquid medium.

Example 5: Effect of controlling Bacillus subtilis EB120 strain against cucumber powdery mildew EB120 was inoculated into trypsin soybean liquid medium and cultured at 30 ° C. and 150 rpm for 3 days. This was diluted with distilled water at ratios of 1:10, 1:25, 1:50, 1: 100 and 1: 200. After adding 250 μg / ml Tween 20 to each diluted solution, the leaf surface of cucumber at the 7th leaf stage was sprayed.

  As a comparative group, commercially available microbial disinfectant “Top Seed” (supplier: Greenbiotech Co. Ltd.) for controlling powdery mildew disease was used at 1:10, 1 using distilled water. : Diluted solution diluted at a ratio of 25, 1:50, 1: 100 and 1: 200 and flusilazole (Flusilazole, purchased from Dongbu Hannong Chemical Co., Ltd.) known as a bacterial agent, as a target cucumber treatment As a control group, cucumber treated only with Tween 20 solution was used.

  One week after the spray treatment, the diluted solution of the strain culture solution and the sample of the comparative group were secondarily treated on the target cucumber, and the powdery mildew by the same method as that described in Example 7 seven days after the second treatment. The disease control effect on was measured. Powdery mildew induced a natural outbreak caused by the pathogen (Sphaerotheca fuligenea) in the greenhouse.

The experimental process was performed twice, and the results of the first and second experiments are shown in Table 4 below.

  As is apparent from Table 4, all the diluted solutions of the EB120 culture solution showed excellent control activity against cucumber powdery mildew, and even when treated with EB120 diluted solution diluted 200 times, it was effective. It was found to show a level of control effect. Moreover, when compared with the top seed which is the microbe disinfectant for the existing powdery mildew control, it turned out that the dilution solution processing group whose dilution factor is 50 times or less shows the effect more than the top seed processing group. FIG. 2 is a diagram showing the control activity of EB120 against cucumber powdery mildew. In the case of the control group treated only with the Tween 20 solution, many symptoms of powdery mildew appeared, whereas in the cucumber leaves of the experimental group treated with the culture solution of Bacillus subtilis EB120, almost all symptoms were found. could not.

Example 6: In vivo test of control activity against plant diseases in Bacillus subtilis EB120 strain culture solution, ethyl acetate and butanol extract thereof To determine the control activity of substances produced by EB120, EB120 culture solution, ethyl acetate thereof And the control effect with respect to the plant disease in butanol extract was experimented as follows.

  EB120 was inoculated into trypsin soybean liquid medium, then cultured in shaking water at 150 rpm for 3 days at 30 ° C., and centrifuged at 9000 rpm for 12 minutes. The supernatant was extracted twice with the same amount of ethyl acetate to obtain an ethyl acetate extract, which was concentrated under reduced pressure. A butanol extract was obtained in the same manner as the ethyl acetate extract. After taking an amount corresponding to 40 ml of the culture solution from each extracted sample, the ethyl acetate extracted sample was dissolved in 4 ml of acetone, and the butanol extracted sample was dissolved in 2 ml of methanol. Both solutions were diluted with Tween 20 (250 μg / ml) solution to make a final volume of 40 ml.

  On the other hand, 250 μg / ml Tween 20 was added to 40 ml of the culture supernatant of EB120.

  Each solution was sprayed onto pepper, rice, tomato, wheat, and barley, and after 1 day, the caustic anthracnose (PAN), rice blast (RCB), rice blast (RSB), tomato gray mold ( TGM), tomato plague (TLB), wheat rust (WLR), and barley powdery mildew (BPM) were inoculated to examine the disease control effect.

  In the pathogenesis induction of each pathogenic fungus, tomato gray mold disease, tomato plague disease, and barley powdery mildew were performed by the same method as described in Example 3.

On the other hand, for pepper anthracnose, seeds of sprouted pepper seeds were sown in a 7.0 cm diameter pot and then grown in a greenhouse until the 8th leaf stage. After spraying a spore suspension (5 × 10 ⁵ spores / ml), the spore suspension was treated in a constant humidity chamber for 2 days and then cultured in a constant temperature and humidity chamber at 25 ° C. for 2 days to induce disease.

Rice blast is inoculated by spraying a 2-leaf stage seedling with a spore suspension (5 × 10 ⁵ spores / ml) of rice blast pathogen (Magnaporthe gisea, available from Seoul National University, Korea) at 25 ° C. After being left in a humid room for 1 day, it was cultured in a thermostatic chamber at 25 ° C. for 5 days to induce disease.

  Rice leaf blight is inoculated into a three-leaf stage seedling with a medium in which rice leaf blight pathogen (Thanatephorus cucumeris, source: Korea Chemical Research Institute) has been cultured for 7 days (90g bran, 15g rice bran, 100ml distilled water). After treatment in a humid chamber at 25 ° C. for 4 days, the disease was induced by culturing in a thermostatic chamber at 25 ° C. for 4 days.

  Wheat red rust is caused by suspending spores of active parasites (Puccinia recondita, available from Korea Chemical Research Institute) in a 1-leaf seedling in a Tween 20 solution (250 μg / ml) at a concentration of 0.67 g spores / l. The spore suspension was sprayed and treated in a humid room at 20 ° C. for one day, and then transferred to a thermostatic chamber and cultured to induce disease.

For pepper anthracnose, 4 days after inoculating the pathogen, for rice blast, 5 days after inoculating the pathogen, for wheat rust and rice blight, inoculated with the pathogen 7 days after that, the lesion area ratio (%) was examined, and the disease control effect (control value) was measured by the method of Example 3. The results are shown in Table 5 below.

  As can be seen from Table 5, the ethyl acetate extract of the EB120 culture solution had little effect on plant diseases, but the butanol extract, like the culture supernatant of the EB120 strain, was barley powdery mildew, It showed high control activity equivalent to or higher than the culture supernatant of EB120 strain against all plant diseases such as pepper anthracnose, rice blast, tomato gray mold, tomato blight, and wheat rust. Accordingly, it was found that the control active substance produced by the EB120 according to the present invention is a substance having a relatively high polarity.

Example 7: Stability survey with respect to temperature of the control active substance produced by Bacillus subtilis EB120 strain To investigate the stability with respect to the temperature of the substance produced by EB120, the culture supernatant and its butanol extract were subjected to various temperatures. That is, after pretreatment at 4 ° C., normal temperature, 30 ° C., 40 ° C., 50 ° C., 60 ° C., 70 ° C., 100 ° C., and 121 ° C., the degree of inhibition of hyphal elongation of the pathogenic strain was investigated by inoculating the pathogenic strain. .

  Specifically, the EB120 culture supernatant and the butanol extract obtained in Example 6 were 24 hours at 4 ° C. and room temperature, 5 hours at 30 ° C., 40 ° C., 50 ° C., 60 ° C., and 70 ° C., Pretreatment was performed at 100 ° C and 121 ° C for 15 minutes. The culture supernatant was diluted with distilled water at a ratio of 1: 2, and the butanol extract was dissolved in methanol at a concentration of 200 mg / ml. 50 μl of each sample was sprayed onto an 8 mm diameter paper disk and dried. A rice blast pathogen (Magnaporthe gisea, available from Seoul National University) was poured, transferred onto a potato agar medium inoculated by the pour-plating method, and cultured at 25 ° C. for 3 days. Subsequently, the hyphal elongation inhibition effect was measured by taking each paper disk and measuring the diameter of the surrounding inhibition zone.

  As shown in FIG. 3, the three areas of other patterns that are affected by the metabolites produced from EB120 in rice blast are as follows. The part where the mycelial growth is completely inhibited (completely inhibited part), the part where the aerial mycelium cannot grow and the mycelium grows inside the medium (partial inhibitory part), the aerial mycelium grows, and the aerial mycelium is adsorbed on the medium There is a part that looks like (the part affected by aerial hyphae). Accordingly, it can be seen that the EB 120 produces at least three or more control active substances.

  Moreover, it is recognized that the control active substance which EB120 produces shows the strain growth inhibitory effect of a substantially uniform grade irrespective of temperature (FIG. 4). Thus, it can be seen that the control active substance produced by EB120 is relatively stable to heat. In FIG. 4, the black bar indicates the complete inhibition site, the gray bar indicates the partial inhibition site, and the white bar indicates the affected area of the aerial mycelium.

  On the other hand, as a result of observing the growth inhibition degree of rice blast pathogen (Magnaporthe gisea) by heat treatment of the butanol extract of the culture solution of Bacillus subtilis EB120 strain, the butanol extract of the culture solution is different from the culture supernatant. The affected area of aerial hyphae did not appear. This result indicates that the control active substance showing the pattern of the affected site of the aerial mycelium is less polar than the control active substance showing two different patterns. FIG. 5 is a graph showing that the butanol extract also exhibits a uniform hyphal elongation inhibitory activity regardless of the heat treatment temperature.

  The preferred embodiments of the present invention have been described in detail above, but the scope of the present invention is not limited to the above-described embodiments, and does not depart from the technical idea of the present invention described in the claims. Obviously, various modifications and changes can be made by those skilled in the art.

It is a photograph of Bacillus subtilis EB120 strain cultured on trypsin soybean agar medium. It is a photograph which shows the antifungal activity with respect to cucumber powdery mildew of Bacillus subtilis EB120 strain. It is a photograph which shows the hyphal elongation inhibitory activity with respect to rice blast fungus of Bacillus subtilis EB120 strain. It is a graph which shows the thermal stability of the culture solution of Bacillus subtilis EB120 strain. It is a graph which shows the thermal stability of the butanol extract of the culture solution of Bacillus subtilis EB120 strain.

Claims (6)

  A biologically pure culture of Bacillus subtilis EB120 strain (KCTC 10578BP) having an antagonistic action against plant diseases.   Bacillus subtilis EB120 strain (KCTC 10578BP) or a substance derived therefrom as an active ingredient, a microbial preparation for controlling plant diseases.   The plant disease control according to claim 2, wherein the substance is selected from the group consisting of an entire culture solution of Bacillus subtilis EB120 strain, a butanol extract of the culture solution, and spores of Bacillus subtilis EB120 strain. Microbial preparation for use.   3. The plant disease according to claim 2, wherein the plant disease is selected from the group consisting of barley powdery mildew, cucumber powdery mildew, pepper pepper anthracnose, rice blast, tomato gray mold, tomato blight, and wheat red rust. Microbial preparation for plant disease control.   A method for controlling plant diseases comprising the step of applying Bacillus subtilis EB120 strain (KCTC 10578BP) or the microbial preparation of claim 2 to a plant in an effective amount.   6. The plant disease according to claim 5, wherein the plant disease is selected from the group consisting of barley powdery mildew, cucumber powdery mildew, chili pepper anthracnose, rice blast, tomato gray mold, tomato blight, and wheat red rust. How to control plant diseases.

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